Phosphonate monoesters are known to be closely related to phosphate diesters which are known to be important components of every living cell. In addition, phosphonate monoesters have heretofore been synthesized, normally by one of two general methods, the first of which includes reaction of a phosphonic dichloride under limiting conditions of alcohol, to reduce the possibility of diester formation, with subsequent hydrolysis of the remaining chloride, and the second of which includes formation of the diester with subsequent hydrolysis to the monoester in aqueous or alcoholic alkali.
With respect to the reaction of a phosphonic dichloride, attention is invited to the work of Fild, M. and Schmutzler, R. appearing in 1972 in Organic Phosphorus Compounds, Volume 4, G. M. Kosolapoff and L. Maier, Ed., Wiley-Interscience, New York, Chapter 8. With respect to reducing the possibility of diester formation, attention is invited to the work of Michaelis, A. and Kammerer, K. appearing in 1875 in Ber., Volume 8, page 1307, and the work of Keay, L. appearing in 1965 in the Canadian Journal of Chemistry, Volume 43, page 2637.
With respect to the formation of the diester, this has been achieved by procedures heretofore described by Kosolapoff, G. M. in 1950 in Organophosphorus Compounds, Wiley, New York, Chapter 7. In addition, methods analogous to the preparation of phosphate triesters have been suggested for formation of the diester (see the work of Cherbuliez, E. appearing in 1973 in Organic Phosphorus Compounds, Volume 6, Wiley-Interscience, New York, Chapter 15), with subsequent hydrolysis to the monoester in aqueous or alcoholic alkali being suggested by Behrman, E. J., Biallas, M. J., Brass, H. J., Edwards, J. O., and Isaks, M., in 1970 in the Journal of Organic Chemistry, Volume 35, page 3063.
Since phosphates are major constitutents of every living cell and constitute the structural basis of the nucleic acids, with phosphate diesters being among the most important chemical bonds known, enzymes which are capable of making or breaking such phosphodiester bonds are of considerable importance. Although many enzymes which hydrolyze phosphodiester bonds are known, those which hydrolyze nucleic acids sequentially from a terminus fit into one of two broad classifications, or types, as observed by Razzell, W. E., in 1967 in Experienta, Volume 23, page 321.
While several methods have been utilized for assaying phosphodiesterase activity, none of the methods have been found to be fully suitable in combining convenience, economy and specificity. This has been due, at least in part, to the fact that the natural nucleotide substrates of these enzymes have properties which change so little on hydrolysis that somewhat sophisticated equipment and techniques are required in order to quantitate the reaction. Properties such as increase in absorbance of uv light, decrease in viscosity, and increase in acid-soluble nucleotides have been used in assays. Synthetic chromogenic esters of nucleotides, such as 4-nitrophenyl esters of 5'-thymidine monophosphate have been employed as phosphodiesterase substrates, with spectrophotometric detection of the 4-nitrophenol produced on hydrolysis, but these materials are too expensive to be widely used. An inexpensive chromogenic phosphate diester, bis-(4-nitrophenyl) phosphate, has been widely used but most phosphodiesterases hydrolyze it very ineffectively, if at all, as reported by Razzell, W. E. and Khorana, H. G. in 1959 in the Journal of Biological Chemistry, Volume 234, page 2105. Thus, a need has existed for a convenient and inexpensive phosphodiesterase substrate.